The determination of salicylate in biological fluids such as human serum, has diagnostic significance. Acetylsalicylic acid (aspirin) is used as an analgesic and as an anti-inflammatory drug for arthritis. It rapidly hydrolyzes to salicylate which has the therapeutic affect and a long half life. The therapeutic level as an analgesic is up to 20 mg/dl. For arthritis the level is up to 30 mg/dl. Problems such as headaches, tinnitus, flushing and hyperventilation occur at higher salicylate levels followed by imbalances in the acid-base level. Salicylate levels above 60 mg/dl can be lethal.
U.S. Pat. No. 4,416,983 discloses a solution method for the determination of salicylate in body serums. The method is based on the following chemical reaction: ##STR1## The method as carried out in U.S. Pat. No. 4,416,983 is a wet assay in which the two chemical reactions listed above are carried out simultaneously in one vessel. That is, all the chemical reagents needed to conduct the reactions are included in a single solution to which the salicylate contained in the sample is added.
That method as disclosed is not adaptable for dry analytical elements. The problem is that elements in which all of the reagents are included in a single layer as taught by U.S. Pat. No. 4,416,983, have poor storage life. Work in our labs has shown that the life of the element as measured by its ability to respond to salicylate in a sample, is very short. The salicylate hydroxylase activity drops upon continued exposure to the hydrazone coupling agent. This problem on the solution thereto is not taught in any of the prior art of which the inventors are aware.